Microsclerotia Production Research Articles

Verticillium Wilt of Redbud in China Caused by Verticillium dahliae.

Chinese redbud (Cercis chinensis Bunge), a member of the Fabaceae, is an important ornamental plant native to China with reported desirable medicinal effects, including stimulating blood circulation, detumescence, and detoxification (1). In October 2011, wilt symptoms of gradual leaf yellowing, wilting, scorching (marginal browning), and twig dieback were observed on plants in Yangling, Shaanxi, China. The incidence of diseased plants was about 20% in two main areas (about 20 ha in Zijingshan Park in Zhengzhou, Henan Province; and about 3,000 ha in Taiping National Forest Park in Xi'an, Shaanxi Province). Wilted leaves of diseased plant senesced and died, but defoliation was not observed. Brown discoloration was observed in vascular tissues of petioles, twigs, and stems of diseased plants, sometimes in a ring pattern. The symptoms were often restricted to the lower part of the tree or a few branches. To identify the causal agent, six twigs (each approximately 50 mm in diameter and 10 cm long) sampled from an infected tree in Yangling were rinsed in running water, surface-sterilized with 75% ethanol for 2 min, rinsed in sterilized water three times, dried, cut into 1 cm long segments, and the segments put onto potato dextrose agar (PDA) medium. A fungal isolate was recovered from diseased vascular tissues of each sample when cultured on PDA in the dark at 25°C. After 5 days, colonies changed from white to black as a result of production of microsclerotia. Microscopic observation revealed that conidiophores were hyaline and verticillate, with three to four phialides at each node. Conidia were ellipsoidal, hyaline, single-celled, and 2.5 to 7.5 × 1.25 to 4.5 μm. On the basis of these morphological characteristics, the fungus was identified as Verticillium dahliae (3). To prove Koch's postulates, the roots of 10 healthy, 1-year-old C. chinensis plants were each irrigated in a greenhouse with 50 ml of a conidial suspension (1.0 × 107 spores/ml) of an isolate recovered from an infected plant (2); five control plants were inoculated similarly with sterilized water. Fifteen days after inoculation, the same wilt symptoms observed on the original plants had developed on 9 of the 10 inoculated plants, whereas the control plants remained healthy. The pathogen was recovered 15 days after inoculation by isolating from petiole and stem tissues of symptomatic plants, but was not isolated from the control plants. The internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of the nuclear ribosomal DNA was PCR-amplified with primers ITS1 and ITS4 (4), and sequenced. BLAST analysis of the ITS sequence (GenBank Accession No. AB735536) showed 100% homology with that of an isolate of V. dahliae (FJ572050). To our knowledge, this is the first report of Verticillium wilt on C. chinensis in China.

Biological control of <i>Macrophomina phaseolina</i> on cowpea (<i>Vigna unguiculata</i>) under dry conditions by bacterial antagonists

Macrophomina phaseolina, the causative agent of charcoal rot of cowpea and many other crops, is a devastating pathogen in many regions worldwide. Single control measures are ineffective or not feasible underfarmers’ conditions. In order to promote biological control as a component of an integrated management approach under arid ecological conditions, 20 bacterial antagonists were isolated from soil samples collectedfrom the rhizosphere of healthy as well as M. Phaseolina-infected cowpea from fields located in the dry savannah zone of West Africa. In dual culture tests with four media, antagonistic activity was variable and depended on the medium used. Growth inhibition was generally good on tryptic soy agar on which two antagonists also inhibited microsclerotia production by M. phaseolina. Effective antagonists were identified as Bacillus subtilis, B. sphaericus and Paenibacillus polymixa. In greenhouse experiments, seed treatment with B. subtilis strain A11 reduced the incidence of M. phaseolina by 89.29% over the untreated control plants, and lower pathogen quantities in plants were confirmed by DAS-ELISA. Bacillus subtilis A11 was rhizosphere competent and maintained high population densities of up to 6 Log10 CFU/g fresh weight on the roots and 5.73 Log10 CFU/g fresh weight on the hypocotyls of cowpea plants over three weeks after inoculation. This antagonist is recommended for seed treatment in combination with other cultural practices for the management of M. phaseolina under arid conditions.Keywords: Bacillus subtilis, charcoal rot, rhizosphere colonization, seed treatment.

Production of microsclerotia of the fungal entomopathogen Lecanicillium lecanii (Hypocreales: Cordycipitaceae) as a biological control agent against soil-dwelling stages of Frankliniella occidentalis (Thysanoptera: Thripidae)

Microsclerotia (MS) production by two isolates of Lecanicillium lecanii on various culture media is described, and the efficacy of MS against western flower thrips is evaluated. High concentrations of MS (2.9–3.1×105·mL−1) were produced in media with C:N ratios of 7.4:1 and 10.3:1 by isolate SN21. Bioassays using soil-incorporated MS resulted in significant infection and mortality of thrips.

An optimized method for in vitro production of Verticillium dahliae microsclerotia

Verticillium dahliae causes wilt disease of many crops worldwide. Microsclerotia are the main resting structure of V. dahliae in soil and can survive for more than 10 years, serving as an important source of primary inoculum. Mass production of microsclerotia in laboratory is valuable for studying various aspects of V. dahilae, such as its biology, epidemiology and control. We failed to produce a sufficient amount of microsclerotia in vitro for one strain of our interest using several previously published methods. Therefore, we developed a new protocol for mass in vitro production of viable microsclerotia. Verticillium strains were cultured in a modified basal agar medium at pH of 11.5 under 20 °C and incubated for 25 days in dark. When 16 strains were subjected to this condition, large numbers of microsclerotia were produced although varied greatly among strains, including the strain that failed to produce microsclerotia with previously published methods. Microsclerotia from 14 of the 16 strains resulted in wilt development on inoculated cotton seedlings.

Development of pilot-scale fermentation and stabilisation processes for the production of microsclerotia of the entomopathogenic fungus Metarhizium brunneum strain F52

Using 100 L stirred-tank bioreactors, we evaluated the effect of fermentation parameters and drying protocols on the production and stabilisation of microsclerotia (MS) of the entomopathogenic fungus Metarhizium brunneum (formerly M. anisopliae F52). Results showed that stirred-tank bioreactors can be used to mass produce stable MS of Metarhizium and that culturing and drying protocols significantly affected MS yield and stability. Length of fermentation (4–7 days) for Metarhizium cultures had no significant impact on biomass accumulation, MS formation or the storage stability of the air-dried MS granules. Although cultures of Metarhizium grown on media with a carbon-to-nitrogen (C:N) ratio of 30:1 produced significantly more biomass when compared to cultures grown in media with a C:N ratio of 50:1, MS formation and desiccation tolerance following drying were similar. After storage for 1 year at 4°C, conidia production by air-dried MS granules from 50:1 media was significantly higher compared to MS granules from 30:1 media. The addition of diatomaceous earth (DE) to cultures of Metarhizium prior to drying at rates of 0–60 g L−1 had no significant effect on MS desiccation tolerance but did impact conidia production. Air-dried MS granules without DE produced significantly more conidia g−1 during the first 4 months of storage, but after 1 year, conidia production was similar regardless of DE content of the MS granule. Microsclerotial granules with higher moisture levels (2.6–5.0% w/w) produced significantly more conidia immediately after drying and MS granules with low moisture (0–2.5% w/w) produced more conidia after 12 months storage.

Effect of culture media on the growth and morphology of Hungarian Macrophomina phaseolina isolates

Macrophomina phaseolina (Tassi) Goidanich can cause serious yield losses in several plant species. Investigations were made on the colony morphology of fifty Macrophomina phaseolina isolates from Hungary, one from Spain and two from Serbia on six different culture media to detect differences between the isolates. The fungus exhibited the most intensive growth on maize-flour agar, followed by the Sabouraud-glucose, maltextract, potato-dextrose, Czapek-Dox and watery agar media. Investigations on the macromorphology of the isolates revealed different extents of aerial mycelia formation and differences in the zonal structure of microsclerotial formation on the individual media. One isolate (Mp 38) turned the Sabouraud-glucose agar medium pale pink. Investigations on the micromorphology of the cultures showed that the isolates also varied with respect to microsclerotial characters (size, shape and production of microsclerotia). The microsclerotia of this fungal pathogen had the greatest diameter (averaging 135.31 μm) on Sabouraud-glucose agar medium. This study was the first in Hungary to prove that Macrophomina phaseolina could be grown on nutrient-deficient watery agar, suggesting that it can tolerate a wide range of ecological circumstances.

Diversity of Macrophomina phaseolina (Tassi) Goid Based on Chlorate Phenotypes and Pathogenicity

Macrophomina phaseolina (Tassi) Goid causes charcoal disease of oilseed plants. In this study 24 isolates, which were obtained from sunflower, soybean and sesame, were compared based on chlorate phenotypes and pathogenicity tests. For chlorate phenotypes, the isolates were grown on potassium chlorate and stored at 30oC in darkness. For pathogenicity test, seeds of sunflower, soybean and maize plants were placed on 6 – day – old colonies of each Macrophomina isolates grown on PDA and kept at 30oC in the dark. Results indicate that the sesame isolates had more colony radius rate on chlorate minimal medium in comparison to the soybean and sunflower isolates. The sesame isolates were chlorate resistant and grew normally with numerous dark microsclerotia production on the potassium chlorate. The soybean and sunflower isolates were chlorate sensitive and divided into two classes. Class 1, include the isolates that grew sparsely with a feathery like pattern, and the other one had a completely restricted radial growth meaning that M. phaseolina isolates differed in their ability to use certain nitrogenous compounds. Analysis of variance showed a significant difference between the colony radius rates of the isolates at 1% probability level. Based on Duncan’s test, the isolates have been divided in 14 classes. Results of pathogenicity test showed that there was significant difference (P< 0.01) between the isolates. The results confirmed that the feathery like pattern of the isolates was more virulent on soybean and sunflower.

First Report on Association of Colletotrichum coccodes with Chili Anthracnose in India.

Chili (Capsicum annuum L.) is an important condiment and cash crop grown throughout India, including Andhra Pradesh, Karnataka, Maharashtra, Punjab, Tamilnadu, and Himachal Pradesh. In Himachal Pradesh (HP), a northwestern Himalayan state of India, chilies including sweet pepper occupy an area of 2,447 ha with total production of approximately 31,810 t and productivity of 13.00 t per hectare. In 2007 and 2008, chili- and sweet pepper-growing areas of HP were surveyed for the prevalence of fruit rot/anthracnose disease caused by a complex of Colletotrichum species. Fields infested with disease were randomly sampled and four samples from each location were collected. Disease incidence ranged from 12.5 to 45.0% based on total plants assessed in the field. Symptoms of disease in the field included light brown, sunken lesions containing salmon-colored masses of conidia and microsclerotia on the fruit. Microscopic examination of the diseased samples revealed a variation in morphology of spores from two isolates (Cc 70 and Cc 74) collected from two locations in HP, the Kotkhai area of district Shimla and Shamsher (Ani) locality of district Kullu. Five fruits and ten leaves from five plants of a susceptible local variety were inoculated with a suspension of 5 × 105 conidia/ml of isolates Cc70 and Cc74 using a pin prick method as described by Montri et al. (2). The inoculated fruits and leaves were kept in humid chambers at 25 ± 1°C with 12 h of light. After 48 h, the fruits and leaves were observed daily for the appearance of disease symptoms. Disease symptoms were similar to those of natural infections but with darker lesions. The fungus was recovered from infected fruit on Mathur's medium (glucose 2.80 g, peptone 2.00 g, magnesium sulfate hydrated 1.72 g, potassium dihydrogen orthophosphate 1.23 g, and agar 1.50 g/liter) and initially produced white-to-gray mycelia that became dark brown with age. Setae were present along with production of microsclerotia by the tenth day of culturing. A daily average growth of 8.1 mm was recorded on potato dextrose agar at 25 ± 1°C. Conidia were hyaline, unicellular, aseptate, and fusiform abruptly tapering to each end, and 15.5 to 19.6 μm long and 4.2 to 5.3 μm wide. The fungus was identified as Colletotrichum coccodes based on morphological and cultural traits as per the descriptions of Junior et al. (1). The identity of the isolates was confirmed by amplifying the internal transcribed spacer (ITS) region using primer pair ITS1 and ITS4 (3). The sequences (550 bp) were subjected to a BLAST search with the isolates showing the highest identity to GenBank Accession Nos. GU935878 and EF017205. The sequences have been submitted to GenBank (Accession Nos. HQ264175 and HQ264176). Very few reports exist about the natural occurrence of C. coccodes on Capsicum spp. around the world. To our knowledge, this current report constitutes the first record of this pathogen on Capsicum spp. from the Indian subcontinent.

Evaluation of Trichoderma harzianum strain T22 as biological control agent of Calonectria pauciramosa

The objective of this research was to evaluate Trichoderma harzianum strain T22 as a biocontrol agent of collar and root rot caused by different Calonectria pauciramosa isolates. Thus, the microsclerotia-forming ability and virulence of twenty C. pauciramosa isolates were assessed. Microsclerotia production varied partially among the isolates and dual culture with T22 on carnation leaf agar revealed isolates with both high and low microsclerotia-forming ability. Inoculation tests on red clover (Triflolium pratense) demonstrated its susceptibility to the pathogen. On red clover, the degree of virulence and T22 effects in controlling infections were highly variable among the isolates tested. A nursery trial performed on Feijoa sellowiana seedlings confirmed previous results, clearly indicating virulence variability among C. pauciramosa isolates. For three isolates tested in nursery trial, T22 effectiveness in controlling infection was inversely related to their degree of virulence. Overall, T. harzianum strain T22 showed good antagonist activity in reducing microsclerotia production on carnation leaf and the incidence and severity of collar and root rot on both selected hosts. This data could be crucial in developing integrated pest management strategies in ornamental plant nurseries.

Disease Severity and Microsclerotium Properties of the Sorghum Sooty Stripe Pathogen, Ramulispora sorghi

Ramulispora sorghi causes sooty stripe of sorghum. Disease severity in irrigated and dryland plots was measured for 25 susceptible sorghum genotypes during the 2007 and 2008 growing seasons using a rating scale based upon percent leaf area infected. Disease severity ratings were approximately 1.4 points higher (P < 0.0001) on the rating scale in the irrigated plots than dryland plots for 2007 and 2008. Sooty stripe lesions were collected from each sorghum genotype in irrigated plots and assessed for mean microsclerotium production within lesions, microsclerotium size, and sporogenic germination, with significant differences apparent between genotypes for microsclerotium size (P = 0.01) and sporogenic germination (P = 0.01). There was no relationship between disease severity and microsclerotium production within leaf lesions, microsclerotium size, or sporogenic germination; however, there was a positive and significant correlation between microsclerotia production within a lesion and microsclerotium size (R2 = 0.19, P < 0.0001). Although microsclerotia from sorghum lesions varied in structural characteristics and their ability to produce spore masses, these qualities were dependent upon the sorghum genotype from which the microsclerotia were derived, because the R. sorghi population was genetically uniform as determined by internal transcribed spacer sequences and random amplified polymorphic DNA polymerase chain reaction.

Two new species of Cladorrhinum

Two new species of Cladorrhinum, C. flexuosum and C. microsclerotigenum, are described and their sequences of the 5.8S and 28S ribosomal DNA and internal transcribed spacers 1 and 2 of the ribosomal RNA gene cluster analyzed. Cladorrhinum flexuosum, isolated from soil in Spain, forms fast-growing, dull yellow colonies, flexuous conidiophores and globose to dacryoid conidia. Cladorrhinum microsclerotigenum, isolated from Musa sp. in Turkey, strongly resembles Cladorrhinum phialophoroides in the production of abundant terminal phialides, ellipsoid conidia and microsclerotia in culture. It differs from C. phialophoroides in its ability to grow at 36 C and high number of intercalary phialides, which only infrequently occur in C. phialophoroides.

The impact of temperature on the production and fitness of microsclerotia of the fungal bioherbicide Mycoleptodiscus terrestris

The impact of growth temperature was evaluated for the fungal plant pathogen Mycoleptodiscus terrestris over a range of temperatures (20–36°C). The effect of temperature on biomass accumulation, colony forming units (cfu), and microsclerotia production was determined. Culture temperatures of 24–30°C produced significantly higher biomass accumulations and 20–24°C resulted in a significantly higher cfu. The growth of M. terrestris was greatly reduced at temperatures above 30°C and was absent at 36°C. The highest microsclerotia concentrations were produced over a wide range of temperatures (20–30°C). These data suggest that a growth temperature of 24°C would optimize the parameters evaluated in this study. In addition to growth parameters, we also evaluated the desiccation tolerance and storage stability of air-dried microsclerotial preparations from these cultures during storage at 4°C. During 5 months storage, there was no significant difference in viability for air-dried microsclerotial preparations from cultures grown at 20–30°C (>72% hyphal germination) or in conidia production (sporogenic germination) for air-dried preparations from cultures grown at 20–32°C. When the effect of temperature on germination by air-dried microsclerotial preparations was evaluated, data showed that temperatures of 22–30°C were optimal for hyphal and sporogenic germination. Air-dried microsclerotial preparations did not germinate hyphally at 36°C or sporogenically at 20, 32, 34, or 36°C. These data show that temperature does impact the growth and germination of M. terrestris and suggest that water temperature may be a critical environmental consideration for the application of air-dried M. terrestris preparations for use in controlling hydrilla.

Temperature response, pathogenicity, seed infection and mutant evaluation against Macrophomina phaseolina causing charcoal rot disease of sesame

Charcoal rot caused by Macrophomina phaseolina is a serious disease of sesame in Pakistan. M. phaseolina sesame isolate was subjected to growth rate test at 10, 15, 20, 25, 30, 35 and 40°C. The optimum temperature for fungal growth and microsclerotia production was found to be 30–35°C. Gray to black, radial fungal colonies with intermediate mycelial growth and jet black oval to round microsclerotia were observed at this optimum range. M. phaseolina was found to be pathogenic against all the 18 tested plant species and this pathogenicity proved its necrophytic behavior. Seed infection efficiency of M. phaseolina was 100% with significant reduction in seed index. For two consecutive years 21 mutants/varieties were screened in the field for their reactions to charcoal rot disease. During 2007 three mutants NS11704S1, NS11304S2 and NS26004 were ranked as resistant while others were moderately resistant to highly susceptible. During 2008 all mutants showed a susceptible to highly susceptible reaction with variable disease reactions. All over screening results revealed that four mutants viz, NS13P1, NS163-1, NS270P1 and NS26004 showed about 50% stand with consistent performance during both years under optimum disease conditions and can be used to manage the disease following the disease management strategies, however in the future improvement for high seed yield along with resistance is a prerequisite for sustainable high production.

A Study of Environmental Factors that Affect Survival of Pumpkin Isolates of Verticillium dahliae

V. dahliae is a destructive soilborne pathogen to many economically important crops worldwide. Knowledge of environmental influences, including temperature, source, and availability of nutrients and pH, on disease development is central to devising control strategies. The effects of root decoction media, pH, and temperature on radial growth and microsclerotia production in 32 Verticillium dahliae (Kleb.) isolates from Trinidad were investigated. There were significant differences (P ≤ 0.05) in colony diameters and growth rates for isolates grown on different media. The highest growth occurred on 5% nutsedge root extract agar (NREA) and the least amount of growth was obtained on 1% pumpkin root extract agar (PREA). Production of microsclerotia was significantly higher on 5% PREA, 5% NREA, and potato dextrose agar (PDA). Growth tests on PDA formulated to different pH levels revealed that the most growth occurred at pH 5.2, but this was not significantly different from colony diameters obtained at pH 4.2, 6.2, and 7.2. pH, however, had a pronounced effect on production of microsclerotia. The optimum temperature for radial growth and formation of microsclerotia in V. dahliae in vitro was found to be 25 ± 1 °C. The effects of isolates and temperature on incidence and symptom severity in susceptible pumpkin plants were also tested under controlled conditions. There was no significant difference in symptom severity when inoculated plants were grown at 25 ± 1 °C and at 30 ± 1 °C. The pathogen was recovered from infected plants grown at 35 ± 1 °C, although growth is completely inhibited at this temperature in vitro. There was no significant difference among isolates in their pathogenic response at the four different temperatures tested.

Roles of the catalytic subunit of cAMP-dependent protein kinase A in virulence and development of the soilborne plant pathogen Verticillium dahliae

Verticillium dahliae is a soilborne fungus that causes vascular wilt disease in a broad range of hosts and survives for many years in the soil in the form of microsclerotia. Although the role of cAMP-dependent protein kinase A (PKA) has been extensively studied in foliar pathogens, there is limited information about its role in soilborne fungal pathogens that infect through the root system. Genome database search revealed the presence of two PKA catalytic subunit genes in V. dahliae, named VdPKAC1 and VdPKAC2. A phylogenetic analysis showed that VdPKAC2 groups with fungal PKA catalytic subunits that appear to play a minor role in PKA activity. This gene was expressed considerably lower than that of VdPKAC1. Although disruption of VdPKAC1 did not affect the ability of V. dahliae to infect through the roots of tomato and eggplant, disease severity was significantly reduced. Since pathogen-derived ethylene is presumed to play a major role in symptom induction in vascular wilt diseases, ethylene generation was measured in fungal culture. The mutants defective in VdPKAC1 produced less ethylene than the corresponding wild type strains, suggesting a regulatory role of PKA in ethylene biosynthesis. Growth rates of these mutants were similar to those of wild type strains, while the rate of spore germination was slightly elevated and conidia production was significantly reduced. When grown on minimal media, the mutants showed greater microsclerotia production compared with the wild type strains. These results suggest multiple roles of VdPKAC1, including virulence, conidiation, microsclerotia formation, and ethylene biosynthesis, in the soilborne fungus V. dahliae.

Production of microsclerotia of the fungal entomopathogen Metarhizium anisopliae and their potential for use as a biocontrol agent for soil-inhabiting insects

Microsclerotia (MS), overwintering structures produced by many plant pathogenic fungi, have not been described for Metarhizium anisopliae. Three strains of M. anisopliae – F52, TM109, and MA1200 – formed MS in shake flask cultures using media with varying carbon concentrations and carbon-to-nitrogen (C:N) ratios. Under the conditions of this study, all strains produced MS, compact hyphal aggregates that become pigmented with culture age, in addition to more typical blastospores and mycelia. While all strains formed desiccation tolerant MS, highest concentrations (2.7–2.9 × 10 8 L −1 liquid medium) were produced in rich media with C:N ratios of 30:1 and 50:1 by strain F52. All three strains of M. anisopliae produced similar biomass concentrations when media and growth time were compared. Strain MA1200 produced higher concentrations of blastospores than the other two strains of M. anisopliae with highest blastospore concentrations (1.6 and 4.2 × 10 8 blastospores ml −1 on days 4 and 8, respectively) in media with the highest carbon and nitrogen concentrations. Microsclerotial preparations of M. anisopliae containing diatomaceous earth survived air-drying (to <5 % moisture) with no significant loss in viability. Rehydration and incubation of air-dried MS granules on water agar plates resulted in hyphal germination and sporogenic germination to produce high concentrations of conidia. Bioassays using soil-incorporated, air-dried MS preparations resulted in significant infection and mortality in larvae of the sugar beet root maggot, Tetanops myopaeformis. This is the first report of the production of sclerotial bodies by M. anisopliae and provides a novel approach for the control of soil-dwelling insects with this entomopathogenic fungus.

Biocontrol of charcoal rot in sorghum by fluorescent pseudomonads associated with the rhizosphere

Charcoal rot of sorghum caused by Macrophomina phaseolina is a disease of economic importance for which a high level of genetic resistance is not available. Therefore, we made an effort to manage this disease with fluorescent pseudomonads associated with field-grown sorghum crops. One hundred and twenty-six fluorescent Pseudomonas spp. from different sorghum growing regions of India were isolated, selected and evaluated for in vitro antifungal activities and three selected strains were further tested for management of charcoal rot under field conditions. Antifungal activities (inhibition of growth, biomass, microsclerotia production, spore germination) of the secondary metabolites and the cell-free culture filtrates of the selected fluorescent pseudomonad strains (SRB129, SRB288 and Pseudomonas chlororaphis SRB127) were studied in detail. SRB127, SRB129 and SRB288 inhibited mycelial growth of M. phaseolina ranging from 30.5 to 76.5% in dual culture assay. The cell-free culture filtrates of these strains at 20% (v/v) concentration significantly reduced the formation and germination of microsclerotia of M. phaseolina. In the field, P. chlororaphis SRB127 emerged as an effective biocontrol agent of charcoal rot of sorghum. The bacterium, when applied as seed treatment, reduced the charcoal rot incidence by >40%, crop-lodging by >20%, and increased grain mass. P. chlororaphis SRB127, when grown in a gnotobiotic sand system, effectively colonized the sorghum root and formed microcolony-like cell-aggregates in some parts of the root. Under glasshouse conditions the bacterium survived in the sorghum rhizosphere without a significant reduction in population. We conclude that the use of a selection of effective strains of bacteria can be a useful component of integrated management of charcoal rot in sorghum.

Effect of crop rotation on specialization and genetic diversity of Macrophomina phaseolina

Charcoal rot, caused by Macrophomina phaseolina, is one of the most important diseases of soybean. Genetic variability among soybean isolates has been observed but the effect of host specialization on genetic variability has not been reported. In this work, isolates from soybean, corn and sunflower were evaluated based on cross inoculations and number of microsclerotia/g of roots. The highest and lowest significant (P<0.005) production of microsclerotia was obtained in soybean (863 microsclerotia/g of roots) and sunflower (578 microsclerotia/g of roots), respectively, regardless of the origin of the isolate. Additionally, the effect of a 20-year crop rotation on genetic variability based on RAPD was studied. Eighty-nine isolates from five populations were obtained from soil samples under four crop rotation systems and an uncropped soil. Seven clusters were obtained considering a similarity level of 85%. Analysis indicated that M. phaseolina is a highly diverse species and also revealed a strong effect of the rotation system on genetic diversity. AMOVA was conducted for the RAPD data. From the total genetic variability, 21% (P<0.0001) could be explained by the differences between populations while 79% could be explained by differences within populations ( Φst = 0.2110; P<0.0001). The mean coefficient of gene differentiation (GST) estimated among the five populations indicated 27% of differentiation between populations similar to the AMOVA results where Φst= 0.2110. Total gene diversity estimated indicated high levels of variability (HT =0.3484). Results suggest that genetic differentiation of M. phaseolina can be altered by crop rotation.

Long-term maintenance ofCylindrocladiumstrains and procedures for inoculum production

Les techniques presentees permettent une conservation a long terme des souches de Cylindrocladium pour les preserver d'importants changements morphologiques et physiologiques. Le principe des methodes appliquees, leurs principaux avantages, le materiel vegetal necessaire, le temps requis et les resultats attendus sont presentes. Materiel et methodes. Le materiel de laboratoire necessaire, ainsi que le detail des neuf etapes utilisees pour la purification monospore de souches de Cylindrocladium, leur conservation a long terme, et la production de conidies et de microsclerotes sont decrits. Resultats. Une fois reactivees, les souches purifiees produisent des colonies qui s'accroissent activement en 48-72 h. De telles colonies peuvent alors etre aisement utilisees pour la production de conidies ou de microsclerotes. (Resume d'auteur)

Effects of no tillage and genetic resistance on sunflower wilt by Verticillium dahliae

From 2001 to 2006 crop seasons three field experiments were run to compare the effect of no tillage (NT) vs. conventional tillage (CT) on Verticillium wilt of sunflower. One experiment had sunflower monocropping (SM) and the others the sequence wheat–sunflower (WS) with 6 years of fescue pasture or seven WS cycles as previous crops. All experimental fields have history of the disease. One cultivar with low resistance was used in SM and two genotype-resistance levels (high and low; characterized by six and eight cultivars each, respectively) in the WS sequences. Leaf mottle severity in all environments, Verticillium dahliae colony forming units (CFU)/g of soil in 2005 or 2006 trials, density of microsclerotia in the stem pith at 0.5 m above the soil line in WS, and grain yield and oil content in WS with fescue as previous crop were recorded. Every year, disease severity was higher in CT than in NT in all trials. In SM, disease severity increased during the 3 years in CT from 58% to 88%, while in NT disease severity remained around the initial level (49%). The V. dahliae-CFU/g of soil after 3 years in SM or three cycles of WS was approximately three times higher in CT than in NT. In WS, density of microsclerotia in stem pith were higher in CT than in NT. In WS with fescue pasture as previous crop, grain yield and oil content tend to increase with NT in relation to CT. The combination of NT and high-resistant genotypes reduced the disease and the production of microsclerotia in stem pith to very low values. NT + high resistance should be viewed as a preventative, not a curative disease management option because the microsclerotia persist in soil for a long time. Therefore, NT + high resistance programs should be initiated early, before inoculum builds up to high levels in the soil. Thus, the combination of NT and high-resistant cultivars promises to be an interesting tool to manage V. dahliae and Verticillium wilt in sunflower and would have potential in other crops like alfalfa, cotton or strawberry.